Calcitonin receptor-like receptor and receptor activity modifying protein 1 in the rat dorsal horn: Localization in glutamatergic presynaptic terminals containing opioids and adrenergic α2C receptors

• Published in: Neuroscience Vol. 148; no. 1; pp. 250 - 265
• Main Authors: Marvizón, J.C.G, Pérez, O.A, Song, B, Chen, W, Bunnett, N.W, Grady, E.F, Todd, A.J
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 10.08.2007; Elsevier
• Subjects: adrenergic receptor; Biological and medical sciences; calcitonin gene-related peptide; CGRP; colocalization; dynorphin; enkephalin; Fundamental and applied biological sciences. Psychology; Neurology; Vertebrates: nervous system and sense organs; dynorphin; NS; phosphate-buffered saline; enkephalin; analysis of variance; calcitonin receptor-like receptor; ANOVA; human embryonic kidney; NK1; VGLUT2; normal serum; VGLUT1; phosphate-buffered saline with 0.5% Triton X-100 and 0.01% thimerosal; PBS; TSA; vesicular glutamate transporter 2; adrenergic receptor; neurokinin 1; calcitonin gene-related peptide; CRLR; colocalization; ROI; HEK; tyramide signal amplification; NA; receptor activity modifying protein 1; dorsal root ganglion; region of interest; DRG; vesicular glutamate transporter 1; PBS/Triton; CGRP; RAMP1; numerical aperture; Spinal cord; Rat; Opioid receptor; Rodentia; Central nervous system; Enkephalin; Calcitonin gene related peptide; Posterior spinal horn; Neuropeptide; Opioid peptide; α2C-adrenergic receptor; Protein; Vertebrata; Mammalia; Calcitonin receptor; Animal; Dynorphin; Localization; Adrenergic receptor; Biological receptor
• ISSN: 0306-4522; 1873-7544
• DOI: 10.1016/j.neuroscience.2007.05.036

Abstract Calcitonin gene-related peptide (CGRP) is abundant in the central terminals of primary afferents. However, the function of CGRP receptors in the spinal cord remains unclear. CGRP receptors are heterodimers of calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1). We studied the localization of CRLR and RAMP1 in the rat dorsal horn using well-characterized antibodies against them, which labeled numerous puncta in laminae I–II. In addition, RAMP1 was found in cell bodies, forming patches at the cell surface. The CRLR- and RAMP1-immunoreactive puncta were further characterized using double and triple labeling. Colocalization was quantified in confocal stacks using Imaris software. CRLR did not colocalize with primary afferent markers, indicating that these puncta were not primary afferent terminals. CRLR- and RAMP1-immunoreactive puncta contained synaptophysin and vesicular glutamate transporter-2 (VGLUT2), showing that they were glutamatergic presynaptic terminals. Electron microscopic immunohistochemistry confirmed that CRLR immunoreactivity was present in axonal boutons that were not in synaptic glomeruli. Using tyramide signal amplification for double labeling with the CRLR and RAMP1 antibodies, we found some clear instances of colocalization of CRLR with RAMP1 in puncta, but their overall colocalization was low. In particular, CRLR was absent from RAMP1-containing cells. Many of the puncta stained for CRLR and RAMP1 were labeled by anti-opioid and anti-enkephalin antibodies. CRLR and, to a lesser extent, RAMP1 also colocalized with adrenergic α2C receptors. Triple label studies demonstrated three-way colocalization of CRLR–VGLUT2–synaptophysin, CRLR–VGLUT2–opioids, and CRLR–opioids–α2C receptors. In conclusion, CRLR is located in glutamatergic presynaptic terminals in the dorsal horn that contain α2C adrenergic receptors and opioids. Some of these terminals contain RAMP1, which may form CGRP receptors with CRLR, but in others CRLR may form other receptors, possibly by dimerizing with RAMP2 or RAMP3. These findings suggest that CGRP or adrenomedullin receptors modulate opioid release in the dorsal horn.